Determination of mammalian glycogen synthase phosphatase activity.

Publisher Summary This chapter describes two GS phosphatase assays, one in which phosphatase activity is determined by 32 P release from 32 P-GS phosphorylated at six different sites and the other, a “coupled assay,” in which phosphatase activity is monitored by its ability to dephosphorylate and activate fully phosphorylated and inactive GS. The first assay allow the determination of the activity of protein phosphatases that dephosphorylate any of the six phosphorylation sites, while the second assay detects activity of phosphatases that act on sites that control GS activity. Although the sensitivity of the first assay can be high, depending on the specific radioactivity of the substrate, detection of a specific phosphatase that acts at distinct sites may be difficult to discern over background dephosphorylation at multiple sites. On the other hand, the “coupled assay” could more readily detect phosphatase activity specific for GS activating sites. For the determination of phosphatase activity by 32 P release from 32 P-labeled GS, the dephosphorylated form of GS is purified, whereas for the ‘‘coupled assay’’ the phosphorylated form is isolated.